Recombinant proteins such as antibodies typically contain a variety of impurities that need to be removed before the protein product is pharmaceutically acceptable. Some of these impurities may include host cell proteins (HCPs), DNA molecules, variant and/or misfolded forms of the product protein, and high molecular weight aggregates (HMWA). The formation of aggregates is problematic during antibody production as it can adversely affect product safety by causing complement activation or anaphylaxis upon administration. Aggregate formation may also hinder manufacturing processes by causing decreased product yield, peak broadening, and loss of activity. These impurities can have a wide range of retention patterns on different modes of chromatography. Removal of such broad spectrum of impurities is often difficult, typically requiring multiple steps involving different modes of chromatography.
Common protein purification methods are predicated on differences in the size, charge, and solubility between the protein to be purified and the contaminants. Protocols based on these parameters include, but are not limited to, affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. These chromatographic methods, however, sometimes present technical difficulties in the separation of aggregated or multimeric species of antibodies. Techniques such as ion exchange and hydrophobic interaction chromatography, for example, may induce the formation of aggregates due to an increased protein concentration or the required changes in buffer concentration and/or pH during elusion. Further, in several instances antibodies show differences in isoelectric points that are too small to allow for their separation by ion-exchange chromatography (Tarditi, J. Immunol. Methods 599:13-20 (1992)). Size exclusion chromatography tends to be cumbersome and results in the significant dilution of the product, which is a hindrance in large-scale, efficiency-based manufacturing processes. Leakage of ligands from affinity chromatography columns can also occur, which results in undesirable contamination of the eluted product (Steindl, J. Immunol. Methods 235:61-69 (2000)).
While several different modalities of chromatography can be employed during the purification of recombinant proteins, the need still exists to develop purification processes that reduce the number of chromatography steps used and that do not destroy, or significantly reduce, the biological activity of the recombinant protein.